[Cell Cycle 3:2, 168-171; February 2004]; 2004 Landes Bioscience

Spotlight on Hypoxia-Inducible Factor

An Unexpected Role for Hypoxic Response

Oxygenation and Inflammation
Carole Peyssonnaux Randall S. Johnson*
Molecular Biology Section; Division of Biological Sciences; University of California, San Diego; San Diego, California USA *Correspondence to: Randall S. Johnson; Division of Biological Sciences; University of California, San Diego; 9500 Gilman Drive, MC-0366; La Jolla, California 920930366 USA; Tel.: 858.822.0509; Fax: 858.822.5833; Email: rjohnson@biomail.ucsd.edu Received 11/01/03; Accepted 12/09/03 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=687

ABSTRACT

KEY WORDS
hypoxia, inflammation, HIF-1

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In tissues injured by infectious processes or trauma, microenvironmental conditions are characterized by low levels of oxygen and nutrients, as well as high concentrations of lactate and reductive metabolites.1,2 In healthy tissues, the oxygen tension is generally between 20 and 70 mmHg (i.e., 2.59% oxygen), whereas inadequate perfusion of diseased tissues can cause the formation of multiple transient or chronic areas of hypoxia, in which oxygen tensions of less than 10 mm Hg (i.e., less than 1% oxygen) have been reported.3 In one of the earliest responses to injury, neutrophils stop flowing inside of the venules and form clamps that can grow to occlude the vessel, reducing blood flow (Fig. 1). In addition, neutrophils cross the vessel wall, migrating into the tissue. The disruption of the blood supply to damaged or inflamed tissues often results in the formation of areas of low oxygen tension in tissues. Low oxygen levels have been described in virtually every site of extensive inflammation,4-8 including sites of cutaneous inflammation, such as skin infections and wounds9 and necrotic tissue foci.10,11 At sites of inflammation, approximately 95% of the myeloid cells are recruited to those sites; thus they need to move against oxygen gradients in order to migrate toward relevant areas of inflammation.3 Thus, myeloid effector cells of the innate immune system such as neutrophils and macrophages have an acute need to respond to these demanding conditions to maintain viability and activity.

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HOW DO THESE MYELOID CELLS GENERATE THE ENERGETIC METABOLISM TO FUNCTION IN A RELATIVELY ANAEROBIC ENVIRONMENT?
Historical studies established that neutrophils and mononuclear phagocytes have evolved a strategy of dependence on glycolysis for ATP production, under both normoxic and hypoxic conditions.12-16 Classical studies with glycolytic inhibitors have also shown that modulating glycolysis inhibits chemotaxis, aggregation, and invasion by macrophages and neutrophils; whereas inhibitors of mitochondrial respiration have little or no effect on these processes.17-20 These efforts demonstrated that, unlike almost all other cells and Cell Cycle 2004; Vol. 3 Issue 2

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THE FUNCTION OF MYELOID CELLS IN A HYPOXIC ENVIRONMENT

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The eradication of invading microorganisms depends initially on innate immunity mechanisms that preexist in all individuals and act within minutes of infection. Pathogen spread is often countered by an inflammatory response that recruits more effector molecules and cells of the innate immune system from local blood vessels, while inducing clotting farther downstream so that pathogens cannot spread throughout the blood. If a microorganism crosses an epithelial barrier and begins to replicate in the tissues of the host, it is, in some cases, immediately recognized by the mononuclear phagocytes, or macrophages, that reside in tissues. Macrophages mature continuously from circulating monocytes that leave the circulation to migrate into tissues throughout the body. The second major family of phagocytes, the neutrophils or polymorphonuclear leukocytes (PMNs) are short-lived cells that are abundant in the blood but are not present in healthy tissues. Both phagocytic cell types play a key role in innate immunity because they can recognize, ingest and destroy many pathogens without the aid of an adaptive immune response. This infiltration of neutrophils and later macrophages to the site of bacterial infection is tightly linked with the need of these immune defense cells to respond to the tissue microenvironment.

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TRANSCRIPTIONAL RESPONSE OF THE HYPOXIC RESPONSE BY HIF-1

The observations described above argued for a pronounced dependence of neutrophils and macrophages on the known functions of the hypoxia inducible transcription factor (HIF-1), one of the principal mediators of adaptation to critically low oxygen levels.22 Numerous lines of investigation have proven that HIF-1 is implicated in most aspects of hypoxia-induced gene expression and is essential for hypoxia-induced increases in glycolysis in normal or diseased tissue.22 HIF-1 is a transcription factor whose expression is regulated by oxygen at the protein level. The HIF-1 heterodimer consists of two helix-loop-helix proteins; these are termed HIF-1, which is the oxygen-responsive component, and HIF-1. The latter, also known as the aryl hydrocarbon receptor nuclear translocator (ARNT), is constitutively expressed. In contrast, HIF-1 is typically only detected under low oxygen concentrations. and is rapidly degraded by the ubiquitin-proteasome pathway under ambient conditions;22 this process is directed by its interaction with the von Hippel-Lindau (VHL) tumor suppressor protein. In response to hypoxia, HIF-1 protein becomes stabilized, due to a cessation of its ubiquitination. HIF-1 accumulates and translocates into nucleus, where it binds to HIF-1, constitutively expressed. The heterodimer HIF-1 binds to the hypoxic response elements (HREs) of target gene regulatory sequences, resulting in the transcription of genes implicated in the control of metabolism and angiogenesis, as well as apoptosis and cellular stress. Some of these direct target genes include glucose transporters, the glycolytic enzymes, erythropoietin and the angiogenic factor vascular endothelial growth factor (VEGF). A central component of the complex regulating HIF-1 turnover is the product of the tumor suppressor gene vhl, encoding the von Hippel-Lindau protein (Maxwell et al., 1999). Mutations of the vhl gene are found in patients suffering from the von Hippel-Lindau disease. Patients with the familial disease are prone to development of malignant tumors at a young age. These tumors show high levels of HIF-1 expression and have pronounced vascular beds with enhanced permeability; these are particularly indicative of high levels of expression of one HIF-1 target, the angiogenic/vascular permeability factor VEGF.23 We and others have shown that HIF-1 positively regulates the expression of at least 30 genes including almost every enzyme in the pathway of anaerobic glycolysis.24 Development of a mammalian model to the study the role of HIF-1 in cellular biology and disease pathogenesis was initially

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tissues, myeloid cells do not typically shift to mitochondrial respiration, even in highly oxygenated environments. Since glycolysis represents the chief means of generating ATP in the absence of oxygen, the reliance of neutrophils and other myeloid cells on this metabolic pathway strongly suggests that they are highly adapted to this hypoxic mode of existence. Neutrophil motility, chemotaxis and aggregation are all fueled by extracellular glucose uptake.21 This reliance on the glycolytic pathway is thus a hallmark of the unique and extravascular mode of existence of these myeloid cells. These classical studies as well as more recent efforts also clearly delineated the functional role of glycolytic ATP production in myeloid cell types, since they show how inhibition of glycolysis and diminution of ATP in these cells prevents normal responses to chemotactic agents and inflammatory stimuli.

HIF-1 IS ESSENTIAL FOR MYELOID CELL-MEDIATED INFLAMMATION

The dependence of macrophages and especially neutrophils on glycolysis for energy generation, together with their localization to hypoxic microenvironments within inflamed tissues, suggested a prominent role for the HIF-1 transcriptional control pathway in myeloid cell biology. Recent investigations had shown that HIF-1 (and its target gene VEGF) were indeed expressed in activated macrophages.28-30 In light of these observations, we performed the first comprehensive analysis of the hypoxic response in myeloid cell mediated inflammation, employing conditional gene targeting in the myeloid cell lineage.27 In this study, we created targeted deletions of the HIF-1, VHL and VEGF via crosses into a background of cre expression driven by the lysozyme M promoter (lysMcre),31 which allows specific deletion of the individual factors in the myeloid lineage. We used these neutrophil and macrophage-specific knockout mice to determine the contribution of HIF-1 in myeloid cell inflammatory responses. We showed that HIF-1 regulates glycolysis in neutrophils and mononuclear phagocytes under both normoxic and hypoxic conditions. We postulated that the large decrease in ATP levels caused by decreased glycolysis in myeloid cells could inhibit or eliminate inflammatory responses in lysMcre/HIF-1 animals. A variety of in vitro and in vivo functional assays of acute and chronic inflammation demonstrated these predictions to be true.27 Functional inactivation of HIF-1 greatly inhibited the cell motility, invasiveness and homotypic adhesion of isolated peritoneal macrophages. In a well-established acute model of cutaneous inflammation, a profound reliance on HIF-1 function was demonstrated for infiltration, edema formation, and tissue destruction caused by granulocytes and macrophages. Deletion of the negative HIF-1 regulator VHL caused a hyperinflammatory response in the same model. Loss of the HIF-1 target gene VEGF eliminated tissue edema but not the other measures of myeloid cell inflammation, indicating that the phenotype resulting from the loss of HIF-1 is not solely due to decreased VEGF expression. Importantly, deletion of HIF-1 causes not only a profound impairment of neutrophils and mononuclear phagocyte function in mediating inflammation but also in bacterial killing.27 These discoveries have spurred interest in the potential for therapeutic modulation of HIF-1 and associated pathways in the treatment of autoimmune disorders such as connective tissue disease and inflammatory bowel disease.32,33 However, as is the case with many pathways of anti-inflammatory therapy, the potential for impairment of essential immune functions and increased susceptibility to infectious agents must be evaluated and weighed. The use of corticosteroids and TNF-neutralizing agents taught this lesson.34 In light of these results, it will be essential to address a comprehensive understanding of the role of HIF1- transcriptional control pathways in the innate immune defense against bacterial pathogens. 169

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hampered by its own essentiality: deletion of the HIF-1 gene in mice was lethal during embryogenesis.25,26 We recently overcame this obstacle by employing conditional gene targeting in the myeloid cell lineage, deleting HIF-1 its target gene VEGF, and its upstream regulator, the von Hippel-Lindau factor (vHL) in separate mouse strains expressing cre recombinase in granulocytes and monocytes/ macrophages. These tissue-specific knockout mice allowed an extensive study of the hypoxic response during inflammation.27

HYPOXIC RESPONSE: OXYGENATION AND INFLAMMATION

A ROLE OF HIF-1 IN BACTERIAL INFECTION?

Further work will be required to evaluate the crucial adaptive function of myeloid cells in mammalian evolution: the innate immune defense against pathogenic microorganisms. The effectiveness of neutrophils and macrophages in innate immune defense against bacterial infection reflects a diverse array of highly specialized cellular functions. First, the myeloid cells must recognize chemotactic signals to engage endothelial receptors, initiate diapedesis, exit the microcirculation and migrate to the tissue focus of infection. Recognition of the pathogen, often enhanced by opsonic factors such as complement, leads to phagocytic uptake of the bacterium within a membrane-bound vesicle (phagosome), which then undergoes acidification. The phagosome fuses with one or more lysosomes to generate a phagolysosomes in which the lysosomal contents are released to destroy the pathogen. The production of reactive oxygen species such as hydrogen peroxide (H202) superoxide anion (02-) and singlet oxygen effect killing of ingested pathogens. The reactive oxygen metabolites are generated by lysosomal NADPH oxidases and other enzymes in a process known as the respiratory burst, as it is accompanied by a transient increase in oxygen consumption. Activation of inducible nitric oxide synthase (iNOS) in myeloid cells can generate nitric oxide (NO) at levels toxic to many bacteria. Myeloid cells also possess granules that contain antimicrobial peptides (e.g., cathelicidins, defensins) and proteases (e.g., elastase, cathepsin) that can be released to effect or enhance microbicidal activity. Finally, the myeloid cells arriving at the site of infection are activated to release proinflammatory cytokines (e.g., IL-8, IL-1, TNF) to amplify the immune response and recruit additional myeloid effector cells, as well as vasoactive factors (e.g., VEGF, and also NO) to increase microvascular permeability and allow extravasation of immune cells and factors to the infected tissues. These multiple functions of the myeloid cell in innate immune defense reflect a need for substantial energy generation to sustain the biosynthesitic capacity, metabolic activation and cytoskeletal rearrangements required to effect microbial killing. These observations argued for a pronounced dependence of neutrophils and macrophages on the known functions of HIF-1. Thus, we can postulate that loss of HIF-1 would exert a pleiotropic effect on the response to bacterial pathogens through the innate immune system. A model pathogen chosen for study of bacterial infection could be a pyogenic bacteria, a leading cause of mucosal and invasive bacterial infection in humans. A cardinal feature of invasive pyogenic bacteria skin and soft tissue infections is development of localized tissue ischemia, thrombosis and necrosis, representing a formidable challenge to the adaptiveness of neutrophils and macrophages in hypoxic microenvironments. It would be essential to determine how does HIF-1 control of the metabolic shift in neutrophils and macrophages affect the innate immune function of these cells in bacterial killing and control of infection, and whether increasing the activity of this pathway can act to promote clearing and resolution of bacterial infection. In conclusion, HIF-1 controls inflammatory response through its regulation of the metabolic switch to glycolysis, a switch that is intrinsic to myeloid cell survival and function. A critical role for the HIF-1 pathway was demonstrated for inflammatory cell recruitment at its very earliest stages27 and is also likely to have a profound effect in the critical initial stages of many medically important bacterial 170

infections. Understanding these events and their application may help in the diagnosis and therapy of several bacterial infectious diseases.
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